I would like to perform biochemical and ultrastructural characterization of a newly recognized sub-component of chromatin that is released from nuclei by mild trypsin treatment. This sub-component consists of circles of 30 and 60 kilobase-pair size classes. Distributions of RNA, DNA and protein in the circles will be determined by enzymatic dissection of ultrastructure which has already been used to establish that the circle backbone is DNA with adherent, RNase sensitive material. Autoradiographic methods and studies involving actinomycin D and alpha-amanitin will determine whether the trypsin released circles function in transcription (which we suspect is the case) or another process. Isolated circles will be examined for DNA sequence complexity which will show whether the circle is a restricted (low complexity) or more general feature of chromatin organization. Specific DNA probes to: globin sequences, integrated adenovirus sequences, and sequences complimentary to nuclear and cytoplasmic RNAs will be used to determine the possible localization of specific sequences in the circle DNA. The manner of trypsin-induced circle released suggests a promising approach, by varying conditions of release, to uncovering new organizational principles of chromatin. A second set of experiments involves exonucleolytic probes of chromatin structure. We have characterized the effects of exonuclease III on chromatin and believe that this enzyme might be used to specifically digest active chromatin. If so, the enzyme would be measuring physical accessibility of chromatin template as an isolated variable. By examining the polarity of exonuclease action we will investigate whether transcription assymetry is inherent in the chromatin or instead, depends on RNA polymerase.